KR900005170B1 - Ant-letrovirul agent - Google Patents

Abstract

The use is new of a protein-polysaccharide (I), produced by hot-water or aq. alkaline soln. extraction of the mycelium or fruiting body obtd. from a culture of a basidiomycete of genus Corioulus, which contains 18-38 wt.% protein and which has a mol. wt. of not less than 5000 (by ultracentrifugal analysis), in prepn. of a medicament for use as an antiretroviral agent. (I) a reverse transcritase inhibitor and inhibits binding of HIV to human lymphocytes, and is eps. useful in treatment of HIV infection, including AIDS.

Description

Antiretroviral drugs

)에 의한 역전사효소 활성 억제비율을 나타내고,First figure is Crestine

Inhibition rate of reverse transcriptase activity by

의 억제활성을 나타냄.2 is KRESTIN for the adsorption of HIV onto lymphocytes.

Inhibitory activity.

The present invention relates to an antiretroviral agent containing as an active ingredient a protein-polysaccharide derived from basidiomycetes belonging to the genus Coriolus.

More specifically, the present invention contains a protein-polysaccharide obtained from the mycelium of the cultured basidiomycetes belonging to the genus Coriolus or hot water or alkaline solution extract of the fruiting body as an active ingredient, wherein the protein-polysaccharide is about 18 to 38% by weight It relates to an antiretroviral drug which contains a protein of and has a molecular weight of 5,000 or more (measured by ultracentrifugation analysis).

Recently, new forms of viral diseases such as hepatitis B, adult T cell leukemia, and AIDS have been of interest. Humans have used vaccination vaccines to fight viral diseases and combat diseases like smallpox, yellow fever and polio. However, wax vaccination is insufficient to treat diseases including the problem of persistent infections or latent infections such as AIDS, and therefore, there has been a need for the development of antiretroviral drugs that have a safe and excellent effect against these diseases.

Several of the inventors have proposed many inventions related to protein-polysaccharides derived from basidiomycetes. Such inventions include U.S. Patent Nos. 4,051,314, 4,202,885, 4,289,688, 4,271,151, 4,202,969, 4,229,570, 4,140,578, 4,237,233, 4,288,555, and 4,162,939, 4,159,225, 4,268,505 and 4,663,438 and British Patents 1,331,513 and 1,581,315.

As a result of our study, when human immunodeficiency virus (hereinafter referred to as HIV) was treated with protein-polysaccharide derived from basidiomycetes belonging to the genus Coriolis, HIV belonging to the retrovirus is human lymphocyte. It has been found that the adsorption to and the protein-polysaccharides have the function of markedly inhibiting the activity of reverse transcriptase essential for viral replication. The present invention has been accomplished based on this finding.

In view of the present invention, there is provided an anti-retroviral agent containing a protein-polysaccharide as an active ingredient produced by extracting a mycelium or fruiting body obtained from a culture of basidiomycetes belonging to the genus Corriolis with hot water or alkaline aqueous solution. Protein-polysaccharides contain about 18-38% by weight of protein and have a molecular weight of greater than 5,000 (as determined by hypercentrifugation analysis).

A feature of the present invention is a protein-polysaccharide obtained from the mycelium of the cultured basidiomycete belonging to the genus Coriolus or from the extract of warm water or alkaline solution of fruiting bodies as an active ingredient, wherein the protein-polysaccharide contains about 18 to 38% by weight of protein It is present in antiretroviral drugs containing and having a molecular weight of 5,000 or more (measured by ultracentrifugation analysis).

Protein-polysaccharides used as active ingredients of pharmaceutical compositions for treating retroviral infectious diseases according to the present invention (antiretroviral drugs) are disclosed in Japanese Patent Publication Nos. 51-36322 (1976) and 56-14274 (1981, United States of America). Corresponds to Patent No. 4,202,969, Nos. 56-14276 (1981, corresponding to US Pat. No. 4,140,578) and No. 56-39288 (1981, corresponding to US Pat. No. 4,268,505), and US Pat. A substance obtainable by the method described in the prior patent such as 4,051,314. Such protein-polysaccharides can be obtained from hot water or alkaline solution extracts of culture broths of mycelium or fruiting bodies of basidiomycetes belonging to the genus Coriolis. This polysaccharide contains about 18 to 38 weight percent protein and has a molecular weight (measured by ultracentrifugation analysis) of at least 5,000, preferably at least 5,000 to 300,000.

Shown below are examples of protein-polysaccharides described in the art:

Protein-polysaccharides in the form of hydrolysates include ninhydrin, anisealdehyde, molybdenum, anthrone, tryptophan-sulfonic acid, cromotoroic acid-sulfonic acid, carbazole cysteine-sulfonic acid, aniline-hydrochloric acid, resorcinol-hydrochloric acid, tolene and thio It is positive for glycol-sulfuric acid reactions, but negative for iron chloride and pelling reactions. The polysaccharide belongs to basidiomycetes, Coriolus versicolor, Coriolus conchifer, Cory Coriolus pargamenus, Coriolus hirsutus, Coriolus biformis, Coriolus consors, and Coriolus fuvessens a method comprising forming a mycelium medium or liquid extract in a fungus selected from the group consisting of pubescens and separating the polysaccharides from the medium or liquid extract It is prepared by the (US Patent No. 4,051,314).

Protein-polysaccharides include Coriolus biformis, Coriolus conchifer, Coriolus consors, Coriolus hirsutus, and Coriolus par Pressure fungus in the genus Coriolis of the basidiomycete polyporacea subfamily selected from the group consisting of Coriolus pargametus, Coriolus pubescens and Coriolus versicolor Contacting with an aqueous solvent at a temperature in the range of 150 ° C. to 200 ° C. to obtain an extract containing the protein-polysaccharide; This extract is purified to remove substances having a molecular weight of 5000 or less; Prepared by separating protein-polysaccharides from this extract (US Pat. No. 4,202,885).

Protein-polysaccharide (a) Coriolus versicolor (Fr.) is extracted with an aqueous alkaline solution having a concentration in the range of 0.01 to 2.0 N at a temperature of 50 ° to 100 ° C; (b) neutralizing the resulting extract and its alkaline salt at a concentration of at least 0.4 M / 1 to make the polysaccharide more spherical in shape; In addition, (c) the spherically modified polysaccharide is manufactured by ultrafiltration through a membrane such as a molecular sieve under a pressure of 0.5 kg / cm 2 or more to remove a mixture having a molecular weight of 5,000 or less (US Pat. No. 4,202,969). number).

Protein-polysaccharides completely excluded from monomers having a molecular weight of 5,000 or less are prepared by extracting nitrogen-containing polysaccharides from fungi belonging to the genus Coriolus of the Polyporaceae family of basidiomycetes at temperatures of 50 ° to 100 ° C, wherein (a) the first extraction step is carried out with water or dilute alkaline aqueous solution; (b) the second extraction step is carried out in an alkaline medium having a degree of definition of about 2.0 or less and above the first extraction step; And (c) the third extraction step is carried out in a prescribed diagram above the second extraction step and below about 2.0 (US Pat. No. 4,229,570).

Protein-polysaccharides in the form of hydrolyzate and having anticancer activity include molybdenum reaction, anthrone-sulfuric acid reaction, tryptophan-sulfuric acid reaction, cysteine-sulfuric acid reaction, aminoguanidine-sulfuric acid reaction, chromotropyric acid-sulfuric acid reaction, carbazocysteine- Positive for sulfuric acid reaction, selivanov reaction, vial reaction, aniline-hydrochloric acid reaction, tolene reaction and thioglycolic acid-sulfuric acid reaction and slightly positive for ninhydrin reaction, the protein-polysaccharide is a fungus belonging to the genus Coriolis Mycelia obtained from artificial cultures of the species are prepared by extraction with an aqueous solvent or water containing a small amount of acid, base or water-soluble organic solvent (Japanese Patent Publication No. 51-36322 (1976)).

A pharmaceutical composition for treating bacterial infections by strains of pathogenic bacterial species resistant to conventional antibiotic treatment is an aqueous solvent containing an antimicrobially effective amount of antibiotics and fungi mycelium belonging to the genus Coriolus of the polyporaceae family of basidiomycetes. Nitrogen-containing polysaccharides having a molecular weight of 5,000 to 300,000, 42.0 to 46.0% carbon, 5.3 to 7.0% hydrogen, 0.5 to 8.0% nitrogen and the remainder as obtained by extraction and determined by ultracentrifugation. of water that is only easily melt acetone, the insoluble oxygen, pyridine, chloroform, and a basic composition of the hexane, in the infrared spectrum at 840㎝ ^-1 and a characteristic absorption band 890㎝ ^-1 and 0˚ to 50˚ [α ] has the specific rotation of the _D ²⁵ 62 as also shown by proton nuclear magnetic resonance spectroscopy: sugar for the protein portion in the three ranges: 38 to 97 It has a ratio of the part, wherein the antibiotic is the nitrogen-containing case be administered in combination with polysaccharide can eradicate the bacterial infectious disease (U.S. Patent No. 4,268,505).

이라는 상표명하에 시판되고 있다("Saikin no Shinyaku"(Latest New Drugs), 28권, 14-16페이지(1977) 및 29권, 96-101페이지(1978) ; "Iyakuhin Yoran"(Handbook of Pharmaceuticals), 1,346페이지, 1979년 5월, 제6판, Yakugyo Jihosha ; 및 "Iryoyaku, Nippon Iyakuhin Shu"(Drugs, A collection of Japanese Pharmacenticals), 제7판, 240페이지(1983) Yakuji Jihosha 참고).Among the protein-polysaccharides according to the present invention, those derived from the mycelium of fungi belonging to the genus Coriolus (FERM-P 2412) (ATCC 20547)) are KRESTIN

(Saikin no Shinyaku "(Latest New Drugs), 28, 14-16 (1977) and 29, 96-101 (1978);" Iyakuhin Yoran "(Handbook of Pharmaceuticals), 1,346, May 1979, 6th Edition, Yakugyo Jihosha; and "Iryoyaku, Nippon Iyakuhin Shu" (Drugs, A collection of Japanese Pharmacenticals), 7th edition, page 240 (1983 Yakuji Jihosha).

)의 특징적 특성을 일별한 것이다.The following description is from KRESTIN

The characteristic characteristics of) are summarized.

The polysaccharide portion of the main fraction consists of β-D-glucan, which has a side chain structure containing 1 → 3, 1 → 4 and 1 → 6 bonds. This polysaccharide contains a protein having a nitrogen content of 3 to 6%. Protein component amino acids are mainly acidic amino acids such as aspartic acid, glutamic acid and neutral amino acids such as valine, leucine and the like. Basic amino acids such as lysine and arginine are present in small amounts. This protein-polysaccharide is soluble in water but hardly soluble in methanol, pyridine, chloroform, benzene and hexane and begins to decompose when heated to about 120 ° C.

은 약 18 내지 38중량 %의 단백질을 함유하며 5,000 내지 300,000의 분자량(초원심분리분석에 의해 측정됨)을 갖는다.KRESTN

Contains about 18 to 38 weight percent protein and has a molecular weight of 5,000 to 300,000 (measured by hypercentrifugation).

은 이미 항암약제로 알려져 있었고, 또 독성이 매우 낮고, 장내균상의 장해를 유발할 가능성이 없어 장기간 투여할 수 있다. 또한, 이것은 변이유발 또는 알레르기 반응에 영향을 미치지 않기때문에 기형 유전작용이나 또는 알레르기 반응을 일이킬 염려가 없어서 의약 용도로 매우 안전한 물질이다.KRESTIN as described above

Is already known as an anticancer drug, and is very low in toxicity and can be administered for a long time because it is unlikely to cause an enterobacterial disorder. In addition, it is a very safe substance for medicinal use because it does not affect the mutagenic or allergic reactions, and thus there is no fear of causing malformation or allergic reactions.

The protein-polysaccharide (hereinafter referred to as "the substance") according to the present invention inhibits the adsorption of human immunodeficiency virus (HIV), a type of retrovirus on human lymphocytes, and is a reverse transcriptase essential for the replication of this virus. Significantly inhibits the activity of.

These facts demonstrate that the material is highly effective in treating retroviral infectious diseases such as AIDS-induced AIDS.

The substance is known to be very safe for organisms because its toxicity is very low and causes little harmful side effects. Acute toxicity of this substance is shown in Table 1 below.

TABLE 1

The acute toxicity shown in the table above was measured by the following test method.

As the test animals, ICR-JCL mice weighing 4 to 5 weeks old and weighing 21 to 24 g and Donlius rats weighing 100 to 150 g weighing 4 to 5 weeks old were used. The material was dissolved in physiological salt solution and administered to test animals via four routes, intravenous, subcutaneous, intraperitoneal, and oral.

General symptoms, lethality and body weight of each test animal were observed over 7 days, after which the test animals were killed and dissected.

As shown in Table 1, even when administered at the maximum possible dose, it was virtually impossible to measure the LD ₅₀ value without causing mortality in rats and mice. This means a high safety of the material for animals.

As can be seen from the above, the substance is useful and safe for use as an antiretroviral drug having a very low acute toxicity and showing an inhibitory effect against retroviral infection. Therefore, this material is effective for the treatment of retroviral infectious diseases.

In using the substance as an antiretroviral agent, the substance can be treated with any form of preparation (pharmaceutical composition) required. In addition, the formulations can be administered by any of the oral or parenteral methods required. The pharmaceutical composition according to the present invention does not reduce its efficiency even when used in combination with other known antiretroviral agents such as azido-3'-dioxythymidine (AZT). Therefore, it can be seen that the use of the substance of the present invention in combination with other known agents such as those described above is an effective method for treating retroviral infectious diseases.

In the case of oral administration, the preparations may take the form of tablets, granules, powders, capsules, and the like, and are generally used for pharmaceutical compositions such as binders, inclusions, excipients, lubricants, disintegrants, wetting agents, or the like. Adjuvants may be included in their compositions. When used as an oral liquid formulation, the compositions of the present invention can be treated with liquids, shake mixtures, suspensions, emulsions, syrups and the like for internal use. The compositions of the invention can also be taken in the form of a dry product which becomes a liquid when used. Such liquid formulations may contain additives and / or preservatives in the form generally used.

In the case of injectables, the compositions of the present invention may contain additives such as stabilizers, buffers, preservatives, isotonic agents and the like, which may be provided in the form of unit dose ampoules or multiple dose inclusions. The compositions of the present invention may be treated in the form of aqueous solutions, suspensions, solutions or emulsions in oily or aqueous excipients. The present material as active ingredient may be in powder form which is redissolved in a suitable excipient such as sterile water without pyrogenic material prior to use.

Antiretroviral agents of the present invention are administered orally or parenterally to humans or animals. Sublingual administration is included in the form of oral administration. Forms of parenteral administration include injection and drip, such as subcutaneous, intramuscular and intravenous injection.

The dosage of the antiretroviral drug of the present invention depends on whether the subject is a human or an animal, and also depends on age, individual difference, disease state, etc., but when the subject is a human, the oral dosage of the substance per 1 kg of body weight per day 10-1000 mg, preferably 20-600 mg, this amount of the substance is administered once or three times.

This substance is extremely safe and effective against HIV-induced retroviral infections such as AIDS (Acquired Immunodeficiency Syndrome).

Although the present invention will be described in more detail with reference to the following examples, these examples are intended to be illustrative and not intended to limit the scope of the invention.

Example 1

Retroviruses have RNA as a single gene and use their reverse transcriptase enzymes in infected cells to synthesize DNA using the RNA gene as a template. Reverse transcriptase is an enzyme specific for retroviruses. In this example, it was examined whether the substance inhibited the action of reverse transcriptase.

200mg을 멸균증류수 10㎖중에 용해시키고 그 용액을 2.0, 1.0, 0.5, 0.2, 0.1 또는 0.05mg/㎖농도로 조정하였다. 20mM D.T.T.(디티오트레이틀, 시그마 컴페니 리미티드 제품) 1ul, 5배농축 효소 반응 용액(250mM 트리스-HCl(pH 8.3), 250mM KCl 및 40mM MgCl₂) 5ul, 3d NTP용액(1mM dATP, 1mM dGTP 및 1mM dTTP, 시그마 컴페니 리미티드 제품) 1ul, 100㎍/㎖ 올리고머(dT)_12-18(PL 바이오케미컬 컴페니 리미티드 제품) 2ul, mRNA(정상 쥐 간으로부터 유도함, 1㎍/㎖) 1ul, RNA 분해효소 억제제(16단위체/ul, 다까라 스조 가부시끼 가이샤 제품) 0.5ul 및 (α-³²P)dCTP(800Ci/m㏖까지, 10μCi/ul, 아미삼 자팬 컴페니 리미티드 제품) 1ul를 1.5㎖ 에펜도르프 튜브에 넣고, 이 에펜도르프 튜브를 37℃ 수욕에 방치하였다.KRESTIN

200 mg were dissolved in 10 ml of sterile distilled water and the solution was adjusted to 2.0, 1.0, 0.5, 0.2, 0.1 or 0.05 mg / ml concentration. 1 ul 20 mM DTT (Dithiotrade, Sigma Company Limited), 5 fold concentrated enzyme reaction solution (250 mM Tris-HCl, pH 8.3), 5 ul 250 mM KCl and 40 mM MgCl ₂ ), 3 d NTP solution (1 mM dATP, 1 mM dGTP) And 1 mM dTTP, Sigma Company Limited) 1 ul, 100 μg / ml oligomer (dT) _12-18 (PL Biochemical Company Limited) 2 ul, mRNA (derived from normal rat liver, 1 μg / ml) 1 ul, RNA 1.5 ml of a degrading enzyme inhibitor (16 units / ul, manufactured by Takara Suzo Co., Ltd.) and 1ul of (α- ³² P) dCTP (up to 800 Ci / mmol, 10 μCi / ul, amisamjapan company limited) It was placed in an Eppendorf tube, and the Eppendorf tube was left in a 37 ° C water bath.

용액 12.5ul를 이 용액 튜브에 첨가하고, 역전사효소(라우스 관련 바이러스로부터 유도됨, 7단위체/ul, 다까라 스조 가부시끼가이샤 제품) 1ul를 더 첨가하여 반응 용액의 최종량이 25ul가 되었고, 이들을 37℃에서 반응시켰다.After 5 minutes, the prepared 1 mg / ml KRESTIN

12.5 ul of solution was added to this solution tube, and 1 ul of reverse transcriptase (derived from Raus-associated virus, 7 units / ul, manufactured by Takarasuzo Co., Ltd.) was added to give a final volume of the reaction solution of 25 ul, 37 The reaction was carried out at 占 폚.

After 1 hour, 5 ul of the reaction solution was infiltrated into a 2 cm x 2 cm DEAE paper sheet (manufactured by Toyo Rossi Co., Ltd.). After air drying, each filter paper sheet was impregnated in 10 ml of 0.5 M Na ₂ HPO ₄ solution and [α- ³² P) dCTP remaining on the filter paper without being used for DNA synthesis was washed off under shaking ( This operation was performed 5 times at 5 minute intervals).

The DEAE paper sheets thus treated are then placed in a glass jar containing 10 ml of liquid scintillation cocktail (probably from Samsam Japan Company Limited) and the radioactivity (cpm) of each DEAE paper sheet is scintillation counter (Aroca Co., Ltd.). Was measured for 1 minute.

용액으로 유사한 시편을 제조하고 이 시편에 대해 상기와 유사한 측정을 실시하였다.2.0, 0.5, 0.2, 0.1 or 0.05 ng / ml KRESTINAN

Similar specimens were prepared from the solution and the similar measurements were made on these specimens.

The percentage of reverse transcriptase inhibition was determined from the following formula.

C _o : Radioactivity of the specimen without addition of this material

C _s : Radioactivity of the specimen to which the present material is added

의 역전사효소 활성 억제 비율을 제1도에 나타낸다.KRESTIN

Figure 1 shows the rate of inhibition of reverse transcriptase activity.

As can be seen in Figure 1, the substance significantly inhibited reverse transcriptase activity.

Example 2

Viruses are adsorbed onto and penetrate target cells and replicate by using such target cells. In this example, it was examined whether the substance inhibited the adsorption of HIV onto human lymphocytes.

용액(0.1, 0.2, 0.4 또는 0.8mg/㎖농도) 각 1㎖를 시험관에 넣고, 이 시험관을 얼음속에 가만히 방치하였다. 2시간 후, 바이러스 현탁액 1㎖를 각 시험관으로부터 취해서 이 바이러스를 사람의 임파구-유래 세포주 MT-4(Jpn. J. Cancer Res. (Gann), 28, 219-229(1982)상에 감염중복도(multiplicity of infection, M.O.I로 약칭) 약 2로 흡착시켰다.1 ml each of human immunodeficiency virus (HIV) suspension and KRESTIN

1 ml of each solution (concentration of 0.1, 0.2, 0.4 or 0.8 mg / ml) was placed in a test tube, and the test tube was left standing in ice. After 2 hours, 1 ml of the virus suspension was taken from each test tube and the virus was infected on human lymphocyte-derived cell line MT-4 (Jpn. J. Cancer Res. (Gann), 28, 219-229 (1982). Adsorbed at about 2 (multiplicity of infection, abbreviated MOI).

After centrifugation at 2,000 rpm for 10 minutes, the supernatant was removed and the precipitated MT-4 cells were lysed in RPMI 1640 (Gibco Laboratories, NY) containing 20% of FCS and the cell concentration was 2 × 10 ⁵ /. It became ml. The MT-4 cell suspension was pipetted and transferred to 100-ul plates in 96-hole plates and incubated in air under conditions of 5% CO ₂ and 37 ° C. On day 3 of culture, HIV adsorbed and non-adsorbed cells were calculated by indirect fluorescent antigen technique.

MT-4 cells were fixed by methanol treatment and reacted with antiserum of HIV-infected patients at 37 ° C. for 30 minutes, then the cells were washed with PBS and further treated with 37 ° C. fluorescein isocyanate-binding rabbit anti-human IgG (immune). Globulin).

500 MT-4 cells were observed under fluorescence microscopy to count fluorescent positive cells as HIV adsorbed cells.

The results are shown in FIG.

As can be seen from Figure 2, the adsorption of HIV onto lymphocytes was completely inhibited by the addition of 400 ul / ml or more of the present material.

As described in Examples 1 and 2, the present substance has not only inhibitory activity but also reverse transcriptase suppression activity against the adsorption of lymphocytes onto lymphocytes, and is considered to be effective against HIV-infected diseases such as AIDS.

Production Example

30mg을 #0 경질 캡슐내에 충전시켜 캡슐을 수득하였다.KRESTIN using a pressure type automatic charger

30 mg were filled into # 0 hard capsules to obtain capsules.

Claims (3)

It contains a protein-polysaccharide obtained from the mycelium of the cultured basidiomycete belonging to the genus Coriolus or from a hot water or alkaline solution extract of the fruiting body, wherein the protein-polysaccharide contains about 18 to 38% by weight of protein and is more than 5,000 Antiretroviral drug with molecular weight (measured by hypercentrifugation analysis). 2. The protein-polysaccharide of claim 1, wherein the protein-polysaccharide is a polysaccharide containing a protein with 3 to 6% nitrogen content, and the polysaccharide portion of the main fraction consists of β-D-glucan, wherein the glucan portion is 1 → 3, 1 → It has the characteristic of having a side chain structure containing 4 and 1 → 6 bonds, wherein the protein component amino acids are mainly acidic amino acids of aspartic acid and glutamic acid and neutral amino acids of valine and leucine, while basic amino acids of lysine and arginine are low in content and polysaccharides. Is soluble in water but hardly soluble in methanol, pyridine, chloroform, benzene and hexane.

Phosphorus antiretroviral agent. The antiretroviral drug of claim 1 wherein the retrovirus is human immunodeficiency virus (HIV).

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